This application claims priority of PCT Application Ser. No. PCT/EP00/01002, filed Feb. 8, 2000 the complete disclosure of which is hereby incorporated by reference.
a) Field of the Invention
The invention is directed to a method and a device for collecting fractions, preferably in microliter scale, and is provided for analytic and preparative applications in biochemistry, molecular biology, chemistry, pharmaceutics and pharmacology, but especially in biotechnology. By way of example, the invention can be used in active ingredient screening in the pharmaceutical industry, clinical chemical analysis, protein analysis after fragmentation and combinatorial chemistry.
b) Description of the Related Art
Over the last decade, methods for material separation, especially in chromatography, have developed considerably and undergone miniaturization. This miniaturization became necessary in particular because frequently only very small substance amounts in the xcexcg range or mg range were available for routine characterization of many biologically active substances. Further, the requirements for specimen throughput in analysis, active ingredient screening, biotechnology and molecular biology have risen sharply in recent years. The level of pump, column and detection technique has made it possible to carry out separation in the range of a few microliters. Recently, in particular, in addition to online tracking of chromatography results, the need has arisen for offline analysis of individual fractions which are collected in high resolution in closely spaced grids (e.g., peptide separations before mass spectrometry: P. L. Courchesne, S. D. Patterson, xe2x80x9cManual microcolumn chromatography for sample cleanup before mass spectrometryxe2x80x9d, Biotechnics 22 (1998), No. 2: 244-250). The required close fractionation results in many individual specimens necessitating an effective parallel processing for purposes of high-resolution characterization.
However, the applied known chromatography separation methods are carried out sequentially and, therefore, for an effective parallel further handling, must be adapted to available grids for multipipetting dispensing and multichannel measurement technique. At present, a commonly used standard for specimen vessels is defined by the multiwell analysis plate, as it is called, and the grid derived from it (for example, N. S. Gerasimova, I. V. Steklova, T. Tuuminen, xe2x80x9cFluorometric method for phenylalanine microplate assay adapted for phenylketonuria screeningxe2x80x9d, Clin. Chem., October 1989 35 (10): 2112-2115; P. D. Matthews, E. T. Wurtzel, xe2x80x9cHigh-throughput microplate format for producing and screening riboprobes from bacterial cellsxe2x80x9d, Biotechniques, June 1995 18 (6): 1000-1002, 1004; P. Wu, S. Daniel-Issakani, K. LaMarco, B. Strulovici, xe2x80x9cAn automated high throughput filtration assay: application to polymerase inhibitor identificationxe2x80x9d,Anal. Biochem., Feb. 15, 1997, 245 (2): 226-230; M. S. Rashed, M. P. Bucknall, D. Little, A. Awad, M. Jacob, M. Alamoudi, M. Alwattar, P. T. Ozand, xe2x80x9cScreening blood spots for inborn errors of metabolism by electrospray tandem mass spectrometry with a microplate batch process and a computer algorithm for automated flagging of abnormal profiles, Clin. Chem., July 1997, 43 (7):1129-1141). The dimensions are determined by the SBS standard. Starting from these dimensions, deriving from a quantity of 96 specimen vessels, there are grid formats with 384, 864 and 1536 specimen vessels (catalogs of Greiner or Corning Costar). Almost all equipment for high specimen throughput in liquid handling and parallel handling is adapted to this grid format and is therefore compatible in the field.
The separating methods with liquid volume-moving separating techniques such as HPLC or FPLC which run sequentially collect the separated specimens with fraction collectors in a determined sequence or with a determined grid either continuously according to volume and time, according to a timed program or according to a predetermined threshold of the respective detector.
Various types of automatic fraction collectors, coupled with liquid separating processes which are often also automated, are known (U.S. Pat. No. 4,422,151; U.S. Pat. No. 4,049,031; or DE 3 520 055). They comprise holders for specimen vessels, a feed for the solution to be collected, and an internal or external control unit. The variants for the arrangement of specimen vessels are as follows: carrousel type (U.S. Pat. No. 3,838,719), spiral (U.S. Pat. No. 3,570,555), rows and columns (U.S. Pat. No. 4,422,151), rows and columns in movable containers (U.S. Pat. No. 4,077,444).
The positioning of the specimen vessels under the outlet opening for the specimen solution is carried out either by a movement of the specimen vessel holder or by a movement of the outlet opening.
The collected volumes are in the lower range of 5 xcexcl (brochure by Pharmacia Biotech: Fraktionssammler am Smart(trademark) System). Formats corresponding to the grid of the multiwell analysis plate are often used for small volumes and high specimen numbers (INTERNET publication: Gilson, for fraction collector FC203, for fraction collector in the combinatorial chromatography system and for the xcexc-fractionator based on a Gilson 221 XL). These fraction collectors have a holding capacity for at least one specimen container. A movable element guides the specimen feed horizontally over the individual specimen vessels fixed in the grid and fills them as prescribed. In addition to the horizontal positioning movement over the respective vessel, there are arrangements which move the feed element, a capillary or capillary tube, vertically into the vessel for depositing the specimen (INTERNET publication by Gilson for the fraction collector in combinatorial chromatography system and the xcexc-fractionator based on a Gilson 221 XL, and by Pharmacia Biotech for the Smart(trademark) System fraction collector). Another vertical movement for separating the last drop at the outlet is patented in DE 4 303 275.
Previous fraction collectors had disadvantages with respect to the small liquid quantities of the specimens, particularly evaporation losses (high surface-to-volume ratio), entrainment contamination between the fractions, airborne contamination such as dust particles and microorganisms, and possible aerosol formation in the collected material.
Reduced evaporation and improved conservation can be achieved by regulating the temperature to below room temperature (Gilson INTERNET publication for thermostatic specimen container in FC206 fraction collector).
Another possibility for reducing evaporation loss and preventing contamination consists in providing the plates with covers by gluing or welding, so that every specimen vessel on the plate is hermetically closed (U.S. Pat. No. 5,056,427; U.S. Pat. No. 5,604,130). Gilson uses a cover for preventing contamination of the collecting vessels in their FC 206 fraction collector. When covering with foil, there is also the possibility of gluing foils manually and of commercially available devices with automatic foil gluing and welding (INTERNET publication: Presto Automated Microplate Sealer by Zymark).
The drawback in all of these covers used for protecting against evaporation and contamination for fraction collector specimens consists in that the openings of the collecting vessels are primarily closed and the covers or foil must therefore be removed before collection and then replaced after collection. First of all, this represents increased labor in specimen handling, i.e., preparatory handling of the collecting vessels and follow-up handling for the collecting process. Second, it is particularly problematic that no sufficient protection is provided during the temporary absence of a closure during fractionation. Further, there is a risk of specimen loss, contamination and faulty fractionation, particularly due to entrainment of fractions, during fractionation. This also renders effective automation of the collecting process impossible. For these reasons, there is essentially no practical use for closure of the collecting vessels.
It is the primary object of the invention to collect fractions in microliter scale without the risk of contamination, liquid losses, mixing up of fractions or aerosol formation and with the lowest possible expenditure on handling for prehandling and posthandling of specimen vessels.
According to the invention, the collecting vessels are closed by a cover which can be perforated, for example, an adhesive foil, or the collecting vessels can already be provided with a hermetic closure of this kind. When positioned, the cover is penetrated by a thin, hollow perforating tip and the fractionated liquid is dispensed through the cover into the selected collecting vessel when the collecting position is reached. The cover prevents the release of aerosol into the environment and seals the collecting vessels so as to protect against evaporation and contamination. Further, it substantially prevents faulty fractionation of substance proportions which, for example, drip onto the multiwell analysis plate into the collecting vessels thereof and are mixed with the collected fraction. After the specimen is dispensed, the hollow needle supplying the specimen moves back into its starting position over the collecting vessel. For reliable prevention of faulty fractionation, the supplying of the liquid to be fractionated to the perforating tip, as substance-dispensing element, is interrupted during the positioning movement of the perforating tip. This interruption is advantageously implemented by an electromechanical valve. The volume which flows in continuously in front of the valve is received by and temporarily stored in an elastic deformation body, preferably a thin elastic feed tube. Residual drops suspended from the perforating tip are deposited into the collecting vessel by wiping off at the vessel bottom or at the surface of the liquid which is to be collected or which has been dispensed and/or by an impulse for expelling the residual solution from the needle tip. The advantageous use of one possibility or the other depends on the specimen to be collected, the collecting vessels to be used and the collecting volume.
During fractionation, the collecting vessels remain closed with the exception of the tiny perforation opening, so that said protection against evaporation loss, contamination, aerosol formation in the environment, etc. is maintained during collection. It is not necessary to provide additional covering for protected conservation, transport and further processing of the fractions in the collecting vessels because, apart from the perforation puncture, the collecting vessels are still substantially covered after fractionation. The expenditure on handling for arranging the thin cover foil which can be perforated, e.g., an adhesive foil, on a multiwell analysis plate is low, and multiwell analysis plates which are covered already during manufacture can be used if required.